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Abstract Background Brassica napus L. (B. napus) is susceptible to waterlogging stress during different cultivation periods. Therefore, it is crucial to enhance the resistance to waterlogging stress to achieve a high and stable yield of B. napus. Results Here we observed significant differences in the responses of two B. napus varieties in root under waterlogging stress. The sensitive variety (23651) exhibited a more pronounced and rapid reduction in cell wall thickness and root integrity compared with the tolerant variety (Santana) under waterlogging stress. By module clustering analysis based on transcriptome data, we identified that cell wall polysaccharide metabolism responded to waterlogging stress in root. It was found that pectin content was significantly reduced in the sensitive variety compared with the tolerant variety. Furthermore, transcriptome analysis revealed that the expression of two homologous genes encoding polygalacturonase-inhibiting protein 2 (PGIP2), involved in polysaccharide metabolic pathways, was highly upregulated in root of the tolerant variety under waterlogging stress. BnaPGIP2s probably confer waterlogging resistance by inhibiting the activity of polygalacturonases (PGs), which in turn reduces the degradation of the pectin backbone polygalacturonic acid. Conclusions Our findings demonstrate that cell wall polysaccharides in root plays a vital role in response to the waterlogging stress and provide a theoretical foundation for breeding waterlogging resistance in B. napus varieties.

Abstract As a Brassica crop, Brassica napus typically has single flowers that contain four petals. The double-flower phenotype of rapeseed has been a desirable trait in China because of its potential commercial value in ornamental tourism. However, few double-flowered germplasms have been documented in B. napus, and knowledge of the underlying genes is limited. Here, B. napus D376 was characterized as a double-flowered strain that presented an average of 10.92 ± 1.40 petals and other normal floral organs. F1, F2 and BC1 populations were constructed by crossing D376 with a single-flowered line reciprocally. Genetic analysis revealed that the double-flower trait was a recessive trait controlled by multiple genes. To identify the key genes controlling the double-flower trait, bulk segregant analysis sequencing (BSA-seq) and RNA-seq analyses were conducted on F2 individual bulks with opposite extreme phenotypes. Through BSA-seq, one candidate interval was mapped at the region of chromosome C05: 14.56–16.17 Mb. GO and KEGG enrichment analyses revealed that the DEGs were significantly enriched in carbohydrate metabolic processes, notably starch and sucrose metabolism. Interestingly, five and thirty-six DEGs associated with floral development were significantly up- and down-regulated, respectively, in the double-flowered plants. A combined analysis of BSA-seq and RNA-seq data revealed that five genes were candidates associated with the double flower trait, and BnaC05.ERS2 was the most promising gene. These findings provide novel insights into the breeding of double-flowered varieties and lay a theoretical foundation for unveiling the molecular mechanisms of floral development in B. napus.

Variation in patterns of recombination in plant genomes provides information about species evolution, genetic diversity and crop improvement. We investigated meiotic crossovers generated in biparental segregating and reciprocal backcross populations of the allopolyploid genome of rapeseed (Brassica napus) (AACC, 2n = 38). A structured set of 1445 intercrossed lines was derived from two homozygous de novo genome-assembled parents that represented the major genetic clusters of semi-winter Chinese and winter European rapeseeds, and was used to increase QTL resolution and achieve genomic reciprocal introgression. A high-density genetic map constructed with 6161 genetic bins and anchored centromere regions was used to establish the pattern of recombination variation in each chromosome. Around 93% of the genome contained crossovers at a mean rate of 3.8 cM Mb−1, with the remaining 7% attributed to centromeres or low marker density. Recombination hotspots predominated in the A genome, including two-thirds of those associated with breeding introgression from B. rapa. Genetic background might affect recombination variation. Introgression of genetic diversity from European winter to Chinese semi-winter rapeseed showed an increase in crossover rate under the semi-winter environment. Evidence for an elevated recombination rate having historically contributed to selective trait improvement includes accumulation of favorable alleles for seed oil content on hotspots of chromosome A10. Conversely, strong artificial selection may affect recombination rate variation, as appears to be the case with a coldspot resulting from strong selection for glucosinolate alleles on A09. But the cold region would be promptly reactivated by crossing design indicated by the pedigree analysis. Knowledge of recombination hotspots and coldspots associated with QTL for 22 traits can guide selection strategies for introgression breeding between the two gene pools. These results and rich genomic resources broaden our understanding of recombination behavior in allopolyploids and may advance rapeseed genetic improvement.

Stem lodging resistance is a serious problem impairing crop yield and quality. ZS11 is an adaptable and stable yielding rapeseed variety with excellent resistance to lodging. However, the mechanism regulating lodging resistance in ZS11 remains unclear. Here, we observed that high stem mechanical strength is the main factor determining the superior lodging resistance of ZS11 through a comparative biology study. Compared with 4D122, ZS11 has higher rind penetrometer resistance (RPR) and stem breaking strength (SBS) at flowering and silique stages. Anatomical analysis shows that ZS11 exhibits thicker xylem layers and denser interfascicular fibrocytes. Analysis of cell wall components suggests that ZS11 possessed more lignin and cellulose during stem secondary development. By comparative transcriptome analysis, we reveal a relatively higher expression of genes required for S-adenosylmethionine (SAM) synthesis, and several key genes (4-COUMATATE-CoA LIGASE, CINNAMOYL-CoA REDUCTASE, CAFFEATE O-METHYLTRANSFERASE, PEROXIDASE) involved in lignin synthesis pathway in ZS11, which support an enhanced lignin biosynthesis ability in the ZS11 stem. Moreover, the difference in cellulose may relate to the significant enrichment of DEGs associated with microtubule-related process and cytoskeleton organization at the flowering stage. Protein interaction network analysis indicate that the preferential expression of several genes, such as LONESOME HIGHWAY (LHW), DNA BINDING WITH ONE FINGERS (DOFs), WUSCHEL HOMEOBOX RELATED 4 (WOX4), are related to vascular development and contribute to denser and thicker lignified cell layers in ZS11. Taken together, our results provide insights into the physiological and molecular regulatory basis for the formation of stem lodging resistance in ZS11, which will greatly promote the application of this superior trait in rapeseed breeding.

Auxin is essential for plant growth and development, also closely related to plant architecture. YUCCA (YUC) proteins catalyse the rate-limiting step for endogenous auxin biosynthesis. In this study, we identified 57 BnYUC genes in Brassica napus (rapeseed) genome, located on 17 chromosomes. Among them, A09 chromosome contained the most BnYUCs as 6. Phylogenetic tree showed that all BnYUC members were divided into 2 evolutionary clusters, including 7 sub-groups. Among the sub-groups, CladeⅠ-C consisted 20 members, evolved independently from Arabidopsis thaliana, and motif 10 was unique. Genomic collinearity analysis showed that YUC collinear gene pairs widely existed in Brassica species, indicating that this family has a high retention rate in the evolution. Expression profile suggested that the BnYUC family genes tended to express in specific growth/development stages as well as suffering stress/hormone treatments. It suggested that dominant expression of specific members existed with each development stage or treatment, indicating that each BnYUC might have a clear division of work, and play a given biological function. This work was expected to build a basis for further study of the involvement of BnYUC in growth, development and stress response.

Abstract Background Heterosis is an important biological phenomenon in which the hybrids exceed the parents in many traits. However, the molecular mechanism underlying seedling heterosis remains unclear. Results In the present study, we analyzed the leaf transcriptomes of strong hybrids (AM, HM) and weak hybrids (CM, HW) and their parents (A, C, H, M, and W) at two periods. Phenotypically, hybrids had obvious biomass heterosis at the seedling stage, with statistically significant differences between the strong and weak hybrids. The transcriptomic analysis demonstrated that the number of differentially expressed genes (DEGs) between parents was the highest. Further analysis showed that most DEGs were biased toward parental expression. The biological processes of the two periods were significantly enriched in the plant hormone signal transduction and photosynthetic pathways. In the plant hormone signaling pathway, DEG expression was high in hybrids, with expression differences between strong and weak hybrids. In addition, DEGs related to cell size were identified. Similar changes were observed during photosynthesis. The enhanced leaf area of hybrids generated an increase in photosynthetic products, which was consistent with the phenotype of the biomass. Weighted gene co-expression network analysis of different hybrids and parents revealed that hub genes in vigorous hybrid were mainly enriched in the plant hormone signal transduction and regulation of plant hormones. Conclusion Plant hormone signaling and photosynthesis pathways, as well as differential expression of plant cell size-related genes, jointly regulate the dynamic changes between strong and weak hybrids and the generation of seedling-stage heterosis. This study may elucidate the molecular mechanism underlying early biomass heterosis and help enhance canola yield.

Chloroplasts are essential for plant growth and development, as they play a key role in photosynthesis. The chloroplast biogenesis process is complex and its regulatory mechanism remains elusive. We characterized a spontaneous Brassica napus (rapeseed) mutant, ytg, that showed a delayed greening phenotype in all green organs and retarded growth. We identified BnaA02.YTG1 encoding a chloroplast-localized tetratricopeptide repeat protein widely expressed in rapeseed tissues. We speculated that the ytg phenotype was caused by the deletion of BnaA02.YTG1 based on sequence comparison of 4608 (with normal green leaves, isolated from the elite Chinese rapeseed cultivar ZS11) and ytg combined with transcriptome data and CRISPR/Cas9 gene editing results. The homologous gene (BnaC02.YTG1) restored the phenotype of the mutant. BnaA02.YTG1 interacted with MORF2, MORF8, and OZ1. RNA editing of the ndhD-2, ndhF-290, petL-5, and ndhG-50 plastid transcripts was affected in ytg. These findings suggested that BnaA02.YTG1 participates in RNA editing events. We predicted 29 RNA editing sites in the chloroplast of Brassica napus by comparison with the Arabidopsis chloroplast genome. We conclude that BnaA02.YTG1 affects the posttranscriptional regulation of plastid gene expression and suggest that a tetratricopeptide repeat protein is involved in the chloroplast RNA editing in rapeseed.

Although utilization of heterosis has largely improved the yield of many crops worldwide, the underlying molecular mechanism of heterosis, particularly for allopolyploids, remains unclear. Here, we compared epigenome and transcriptome data of an elite hybrid and its parental lines in three assessed tissues (seedling, flower bud, and silique) to explore their contribution to heterosis in allopolyploid B. napus. Transcriptome analysis illustrated that a small proportion of non-additive genes in the hybrid compared with its parents, as well as parental expression level dominance, might have a significant effect on heterosis. We identified histone modification (H3K4me3 and H3K27me3) variation between the parents and hybrid, most of which resulted from the differences between parents. H3K4me3 variations were positively correlated with gene expression differences among the hybrid and its parents. Furthermore, H3K4me3 and H3K27me3 were rather stable in hybridization and were mainly inherited additively in the B. napus hybrid. Together, our data revealed that transcriptome reprogramming and histone modification remodeling in the hybrid could serve as valuable resources for better understanding heterosis in allopolyploid crops.

Flowering time is strongly related to the environment, while the genotype-by-environment interaction study for flowering time is lacking in Brassica napus. Here, a total of 11,700,689 single nucleotide polymorphisms in 490 B. napus accessions were used to associate with the flowering time and related climatic index in eight environments using a compressed variance-component mixed model, 3VmrMLM. As a result, 19 stable main-effect quantitative trait nucleotides (QTNs) and 32 QTN-by-environment interactions (QEIs) for flowering time were detected. Four windows of daily average temperature and precipitation were found to be climatic factors highly correlated with flowering time. Ten main-effect QTNs were found to be associated with these flowering-time-related climatic indexes. Using differentially expressed gene (DEG) analysis in semi-winter and spring oilseed rapes, 5,850 and 5,511 DEGs were found to be significantly expressed before and after vernalization. Twelve and 14 DEGs, including 7 and 9 known homologs in Arabidopsis, were found to be candidate genes for stable QTNs and QEIs for flowering time, respectively. Five DEGs were found to be candidate genes for main-effect QTNs for flowering-time-related climatic index. These candidate genes, such as BnaFLCs, BnaFTs, BnaA02.VIN3, and BnaC09.PRR7, were further validated by the haplotype, selective sweep, and co-expression networks analysis. The candidate genes identified in this study will be helpful to breed B. napus varieties adapted to particular environments with optimized flowering time.

Abstract Background In terms of global demand, rapeseed is the third-largest oilseed crop after soybeans and palm, which produces vegetable oil for human consumption and biofuel for industrial production. Roots are vital organs for plant to absorb water and attain mineral nutrients, thus they are of great importance to plant productivity. However, the genetic mechanisms regulating root development in rapeseed remain unclear. In the present study, seven root-related traits and shoot biomass traits in 280 Brassica napus accessions at five continuous vegetative stages were measured to establish the genetic basis of root growth in rapeseed. Results The persistent and stage-specific genetic mechanisms were revealed by root dynamic analysis. Sixteen persistent and 32 stage-specific quantitative trait loci (QTL) clusters were identified through genome-wide association study (GWAS). Root samples with contrasting (slow and fast) growth rates throughout the investigated stages and those with obvious stage-specific changes in growth rates were subjected to transcriptome analysis. A total of 367 differentially expressed genes (DEGs) with persistent differential expressions throughout root development were identified, and these DEGs were significantly enriched in GO terms, such as energy metabolism and response to biotic or abiotic stress. Totally, 485 stage-specific DEGs with different expressions at specific stage were identified, and these DEGs were enriched in GO terms, such as nitrogen metabolism. Four candidate genes were identified as key persistent genetic factors and eight as stage-specific ones by integrating GWAS, weighted gene co-expression network analysis (WGCNA), and differential expression analysis. These candidate genes were speculated to regulate root system development, and they were less than 100 kb away from peak SNPs of QTL clusters. The homologs of three genes (BnaA03g52990D, BnaA06g37280D, and BnaA09g07580D) out of 12 candidate genes have been reported to regulate root development in previous studies. Conclusions Sixteen QTL clusters and four candidate genes controlling persistently root development, and 32 QTL clusters and eight candidate genes stage-specifically regulating root growth in rapeseed were detected in this study. Our results provide new insights into the temporal genetic mechanisms of root growth by identifying key candidate QTL/genes in rapeseed.

Inheritable albino mutants are excellent models for exploring the mechanism of chloroplast biogenesis and development. However, only a few non-lethal albino mutations have been reported to date in Brassica species. Here, we describe a resynthesized Brassica napus mutant, whose leaf, stem, and silique tissues showed an inheritable albino phenotype under field conditions after the bud stage but green phenotype in the greenhouse during the whole growing season, indicating that the albino phenotype depends on environmental conditions. Compared with the green leaves of the field-grown wild-type (GL) and greenhouse-grown mutant (WGL) plants, white leaves of the field-grown mutant (WL) showed significantly lower chlorophyll contents and structural defects in chloroplasts. Genetic analysis revealed that the albino phenotype of WL is recessive and is controlled by multiple genes. Bulk segregant analysis-sequencing (BSA-Seq) indicated that the candidate regions responsible for the albino phenotype spanned a total physical distance of approximately 49.68 Mb on chromosomes A03, A07, A08, C03, C04, C06, and C07. To gain insights into the molecular mechanisms that control chloroplast development in B. napus, we performed transcriptome (RNA-Seq) analysis of GL, WGL, and WL samples. GO and KEGG enrichment analyses suggested that differentially expressed genes (DEGs) associated with leaf color were significantly enriched in photosynthesis, ribosome biogenesis and chlorophyll metabolism. Further analysis indicated that DEGs involved in chloroplast development and chlorophyll metabolism were likely the main factors responsible for the albino phenotype in B. napus. A total of 59 DEGs were screened in the candidate regions, and four DEGs (BnaC03G0522600NO, BnaC07G0481600NO, BnaC07G0497800NO, and BnaA08G0016300NO) were identified as the most likely candidates responsible for the albino phenotype. Altogether, this study provides clues for elucidating the molecular mechanisms underlying chloroplast development in B. napus.

Abstract Background Cytoplasmic male sterility (CMS), which naturally exists in higher plants, is a useful mechanism for analyzing nuclear and mitochondrial genome functions and identifying the role of mitochondrial genes in the plant growth and development. Polima (pol) CMS is the most universally valued male sterility type in oil-seed rape. Previous studies have described the pol CMS restorer gene Rfp and the sterility-inducing gene orf224 in oil-seed rape, located in mitochondria. However, the mechanism of fertility restoration and infertility remains unknown. Moreover, it is still unknown how the fecundity restorer gene interferes with the sterility gene, provokes the sterility gene to lose its function, and leads to fertility restoration. Result In this study, we used multi-omics joint analysis to discover candidate genes that interact with the sterility gene orf224 and the restorer gene Rfp of pol CMS to provide theoretical support for the occurrence and restoration mechanisms of sterility. Via multi-omics analysis, we screened 24 differential genes encoding proteins related to RNA editing, respiratory electron transport chain, anther development, energy transport, tapetum development, and oxidative phosphorylation. Using a yeast two-hybrid assay, we obtained a total of seven Rfp interaction proteins, with orf224 protein covering five interaction proteins. Conclusions We propose that Rfp and its interacting protein cleave the transcript of atp6/orf224, causing the infertility gene to lose its function and restore fertility. When Rfp is not cleaved, orf224 poisons the tapetum cells and anther development-related proteins, resulting in pol CMS mitochondrial dysfunction and male infertility. The data from the joint analysis of multiple omics provided information on pol CMS’s potential molecular mechanism and will help breed B. napus hybrids.

Chlorophyll biosynthesis and chloroplast development are crucial to photosynthesis and plant growth, but their regulatory mechanism remains elusive in many crop species. We isolated a Brassica napus yellow-virescent leaf (yvl) mutant, which exhibited yellow-younger-leaf and virescent-older-leaf with decreased chlorophyll accumulation and delayed chloroplast development. We mapped yvl locus to a 70-kb interval between molecular markers yvl-O10 and InDel-O6 on chromosome A03 in BC2F2 population using whole genome re-sequencing and bulked segregant analysis. The mutant had a ‘C’ to ‘T’ substitution in the coding sequence of BnaA03.CHLH, which encodes putative H subunit of Mg-protoporphyrin IX chelatase (CHLH). The mutation resulted in an imperfect protein structure and reduced activity of CHLH. It also hampered the plastid encoded RNA polymerase which transcribes regulatory genes of photosystem II and I. Consequently, the chlorophyll a/b and carotenoid contents were reduced and the chloroplast ultrastructure was degraded in yvl mutant. These results explain that a single nucleotide mutation in BnaA03.CHLH impairs PEP activity to disrupt chloroplast development and chlorophyll biosynthesis in B. napus.

Brassica napus L. (canola, oil seed rape) is one of the world’s most important oil seed crops. In the last four decades, the discovery of cytoplasmic male-sterility (CMS) systems and the restoration of fertility (Rf) genes in B. napus has improved the crop traits by heterosis. The homologs of Rf genes, known as the restoration of fertility-like (RFL) genes, have also gained importance because of their similarities with Rf genes. Such as a high non-synonymous/synonymous codon replacement ratio (dN/dS), autonomous gene duplications, and a possible engrossment in fertility restoration. B. napus contains 53 RFL genes on chromosomes A9 and C8. Our research aims to study the function of BnaRFL11 in fertility restoration using the CRISPR/Cas9 genome editing technique. A total of 88/108 (81.48%) T0 lines, and for T1, 110/145 (75%) lines carried T-DNA insertions. Stable mutations were detected in the T0 and T1 generations, with an average allelic mutation transmission rate of 81%. We used CRISPR-P software to detect off-target 50 plants sequenced from the T0 generation that showed no off-target mutation, signifying that if the designed sgRNA is specific for the target, the off-target effects are negligible. We also concluded that the mutagenic competence of the designed sgRNAs mediated by U6-26 and U6-29 ranged widely from 31% to 96%. The phenotypic analysis of bnarfl11 revealed defects in the floral structure, leaf size, branch number, and seed production. We discovered a significant difference between the sterile line and fertile line flower development after using a stereomicroscope and scanning electron microscope. The pollen visibility test showed that the pollen grain had utterly degenerated. The cytological observations of homozygous mutant plants showed an anther abortion stage similar to nap-CMS, with a Orf222, Orf139, Ap3, and nad5c gene upregulation. The bnarfl11 shows vegetative defects, including fewer branches and a reduced leaf size, suggesting that PPR-encoding genes are essential for the plants’ vegetative and reproductive growth. Our results demonstrated that BnaRFL11 has a possible role in fertility restoration. The current study’s findings suggest that CRISPR/Cas9 mutations may divulge the functions of genes in polyploid species and provide agronomically desirable traits through a targeted mutation.

Rapeseed (Brassica napus L.) is one of the most important oil crops in the world. The planting area and output of rapeseed are affected by the flowering time, which is a critical agronomic feature. COL9 controls growth and development in many different plant species as a member of the zinc finger transcription factor family. However, BnaCOL9 in rapeseed has not been documented. The aim of this study was to apply CRISPR/Cas9 technology to create an early-flowering germplasm resource to provide useful material for improving the early-maturing breeding of rapeseed. We identified four COL9 homologs in rapeseed that were distributed on chromosomes A05, C05, A03, and C03. We successfully created quadruple BnaCOL9 mutations in rapeseed using the CRISPR/Cas9 platform. The quadruple mutants of BnaCOL9 flowered earlier than the wild-type. On the other hand, the flowering time of the BnaCOL9 overexpression lines was delayed. An analysis of the expression patterns revealed that these genes were substantially expressed in the leaves and flowers. A subcellular localization experiment demonstrated that BnaCOL9 was in the nucleus. Furthermore, we discovered that two key flowering-related genes, BnaCO and BnaFT, were highly elevated in the BnaCOL9 mutants, but dramatically downregulated in the BnaCOL9 overexpression lines. Our findings demonstrate that BnaCOL9 is a significant flowering inhibitor in rapeseed and may be employed as a crucial gene for early-maturing breeding.

AbstractRapeseed is a globally cultivated commercial crop, primarily grown for its oil. High-density single nucleotide polymorphism (SNP) arrays are widely used as a standard genotyping tool for rapeseed research, including for gene mapping, genome-wide association studies, germplasm resource analysis, and cluster analysis. Although considerable rapeseed genome sequencing data have been released, DNA arrays are still an attractive choice for providing additional genetic data in an era of high-throughput whole-genome sequencing. Here, we integrated re-sequencing DNA array data (32,216, 304 SNPs) from 505 inbred rapeseed lines, allowing us to develop a sensitive and efficient genotyping DNA array, Bnapus50K, with a more consistent genetic and physical distribution of probes. A total of 42,090 high-quality probes were filtered and synthesized, with an average distance between adjacent SNPs of 8 kb. To improve the practical application potential of this array in rapeseed breeding, we also added 1,618 functional probes related to important agronomic traits such as oil content, disease resistance, male sterility, and flowering time. The additional probes also included those specifically for detecting genetically modified material. These probes show a good detection efficiency and are therefore useful for gene mapping, along with crop variety improvement and identification. The novel Bnapus50K DNA array developed in this study could prove to be a quick and versatile genotyping tool for Bnapus

Oilseed rape (Brassica napus) has significant heterosis and Ogura CMS is a major way to use it. Ogura CMS has the advantages of complete and stable male sterility and easy-to-breed maintainers. Therefore, to breed better restorers has become an important goal for this system. Incidentally, clubroot is a soil-borne disease that is difficult to control by fungicidal chemicals, and it has been the main disease of oilseed rape in recent years in China, severely restricting the development of the oilseed rape industry. At present, the most effective method for controlling clubroot disease is to cultivate resistant varieties. One Ogura CMS restorer line (R2163) has shown much better combining ability, but lacks the clubroot disease resistance. This study was carried out to improve R2163 through marker-assisted backcross breeding (MABB). The resistant locus PbBa8.1 was introduced into the restorer R2163, and we then selected R2163R with clubroot disease resistance. Using the new restorer R2163R as the male parent and the sterile lines 116A and Z11A as the female parent, the improved, new resistant hybrids Kenyouza 741R and Huayouza 706R performed well, providing strong resistance and good agronomic traits. This work advances the utilization of heterosis and breeding for clubroot disease resistance in B. napus.

Abstract Background Alloplasmic lines, in which the nuclear genome is combined with wild cytoplasm, are often characterized by cytoplasmic male sterility (CMS), regardless of whether it was derived from sexual or somatic hybridization with wild relatives. In this study, we sequenced and analyzed the mitochondrial genomes of five such alloplasmic lines in Brassica juncea. Results The assembled and annotated mitochondrial genomes of the five alloplasmic lines were found to have virtually identical gene contents. They preserved most of the ancestral mitochondrial segments, and the same candidate male sterility gene (orf108) was found harbored in mitotype-specific sequences. We also detected promiscuous sequences of chloroplast origin that were conserved among plants of the Brassicaceae, and found the RNA editing profiles to vary across the five mitochondrial genomes. Conclusions On the basis of our characterization of the genetic nature of five alloplasmic mitochondrial genomes, we speculated that the putative candidate male sterility gene orf108 may not be responsible for the CMS observed in Brassica oxyrrhina and Diplotaxis catholica. Furthermore, we propose the potential coincidence of CMS in alloplasmic lines. Our findings lay the foundation for further elucidation of male sterility gene.

As a desirable agricultural trait, multi-inflorescence (MI) fulfills the requirement of mechanized harvesting and yield increase in rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait remain poorly understood. We previously identified a difference of one pair of dominant genes between the two mapping parental materials. In this study, phenotype and expression analysis indicated that the imbalance of the CLAVATA (CLV)-WUSCHEL (WUS) feedback loop may contribute to the abnormal development of the shoot apical meristem (SAM). BnaMI was fine-mapped to a 55 kb genomic region combining with genotype and phenotype of 5768 BCF1 individuals using a traditional mapping approach. Through comparative and expression analyses, combined with the annotation in Arabidopsis, five genes in this interval were identified as candidate genes. The present findings may provide assistance in functional analysis of the mechanism associated with multi-inflorescence and yield increase in rapeseed.

Leaf size is a crucial component of sesame (Sesamum indicum L.) plant architecture and further influences yield potential. Despite that it is well known that leaf size traits are quantitative traits controlled by large numbers of genes, quantitative trait loci (QTL) and candidate genes for sesame leaf size remain poorly understood. In the present study, we combined the QTL-seq approach and SSR marker mapping to identify the candidate genomic regions harboring QTL controlling leaf size traits in an RIL population derived from a cross between sesame varieties Zhongzhi No. 13 (with big leaves) and ZZM2289 (with small leaves). The QTL mapping revealed 56 QTL with phenotypic variation explained (PVE) from 1.87 to 27.50% for the length and width of leaves at the 1/3 and 1/2 positions of plant height. qLS15-1, a major and environmentally stable pleiotropic locus for both leaf length and width explaining 5.81 to 27.50% phenotypic variation, was located on LG15 within a 408-Kb physical genomic region flanked by the markers ZMM6185 and ZMM6206. In this region, a combination of transcriptome analysis with gene annotations revealed three candidate genes SIN_1004875, SIN_1004882, and SIN_1004883 associated with leaf growth and development in sesame. These findings provided insight into the genetic characteristics and variability for sesame leaf and set up the foundation for future genomic studies on sesame leaves and will serve as gene resources for improvement of sesame plant architecture.

Plant architecture involves important agronomic traits affecting crop yield, resistance to lodging, and fitness for mechanical harvesting in Brassica napus. Breeding high-yield varieties with plant architecture suitable for mechanical harvesting is the main goal of rapeseed breeders. Here, we report an accession of B. napus (4942C-5), which has a dwarf and compact plant architecture in contrast to cultivated varieties. A BC8 population was constructed by crossing a normal plant architecture line, 8008, with the recurrent parent 4942C-5. To investigate the molecular mechanisms underlying plant architecture, we performed phytohormone profiling, bulk segregant analysis sequencing (BSA-Seq), and RNA sequencing (RNA-Seq) in BC8 plants with contrasting plant architecture. Genetic analysis indicated the plant architecture traits of 4942C-5 were recessive traits controlled by multiple genes. The content of auxin (IAA), gibberellin (GA), and abscisic acid (ABA) differed significantly between plants with contrasting plant architecture in the BC8 population. Based on BSA-Seq analysis, we identified five candidate intervals on chromosome A01, namely those of 0 to 6.33 Mb, 6.45 to 6.48 Mb, 6.51 to 6.53 Mb, 6.77 to 6.79 Mb, and 7 to 7.01 Mb regions. The RNA-Seq analysis revealed a total of 4378 differentially expressed genes (DEGs), of which 2801 were up-regulated and 1577 were down-regulated. There, further analysis showed that genes involved in plant hormone biosynthesis and signal transduction, cell structure, and the phenylpropanoid pathway might play a pivotal role in the morphogenesis of plant architecture. Association analysis of BSA-Seq and RNA-Seq suggested that seven DEGs involved in plant hormone signal transduction and a WUSCHEL-related homeobox (WOX) gene (BnaA01g01910D) might be candidate genes responsible for the dwarf and compact phenotype in 4942C-5. These findings provide a foundation for elucidating the mechanisms underlying rapeseed plant architecture and should contribute to breed new varieties suitable for mechanization.

Brassica napus (oilseed rape) is one of the most important oil crops worldwide, but its growth is seriously threatened by Sclerotinia sclerotiorum. The mechanism of oilseed rape response to this pathogen has rarely been studied. Here, it was identified that BnaA03.MKK5 whose expression was induced by S. sclerotiorum infection was involved in plant immunity. BnaA03.MKK5 overexpression lines exhibited decreased disease symptoms compared to wild-type plants, accompanied by the increased expression of camalexin-biosynthesis-related genes, including BnPAD3 and BnCYP71A13. In addition, two copies of BnMPK3 (BnA06.MPK3 and BnC03.MPK3) were induced by Sclerotinia incubation, and BnaA03.MKK5 interacted with BnaA06.MPK3/BnaC03.MPK3 in yeast. These interactions were confirmed using in vivo co-immunoprecipitation assays. In vitro phosphorylation assays showed that BnaA06.MPK3 and BnaC03.MPK3 were the direct phosphorylation substrates of BnaA03.MKK5. The transgenic oilseed rape plants including BnaA06.MPK3 and BnaC03.MPK3 overexpression lines and BnMPK3 gene editing lines mediated by CRISPR/Cas9 were generated; the results of the genetic transformation of BnaA06.MPK3/BnaC03.MPK3 indicate that BnMPK3 also has a positive role in Sclerotinia resistance. This study provides information about the potential mechanism of B. napus defense against S. Sclerotiorum mediated by a detailed BnaA03.MKK5-BnaA06.MPK3/BnaC03.MPK3 module.

Drought is a major threat to plant growth and crop productivity. Reduced level of the gibberellin would result in increased drought tolerance, but the underlying mechanism is still unclear. In Brassica napus, there are four BnaRGA genes that code for DELLA proteins, negative regulators of GA signaling. Among them, expression of BnaA6.RGA was greatly induced by drought and abscisic acid (ABA). Previously, we created the gain-of-function mutant of BnaA6.RGA, bnaa6.rga-D, and the loss-of-function quadruple mutant, bnarga by CRISPR/Cas9, respectively. Here we show that bnaa6.rga-D displayed enhanced drought tolerance, and its stomatal closure was hypersensitive to ABA treatment. By contrast, bnarga displayed reduced drought tolerance and was less sensitive to ABA treatment, but there is no difference in drought tolerance between single BnaRGA mutant and WT, suggesting a functional redundancy between the BnaRGA genes in this process. Furthermore, we found that BnaRGAs were able to interact physically with BnaA10.ABF2, an essential transcription factor in ABA signaling. The BnaA10.ABF2-BnaA6.RGA protein complex greatly increased the expression level of the drought responsive gene BnaC9.RAB18. Taken together, this work highlighted the fundamental roles of DELLA proteins in drought tolerance in B. napus, and provide desirable germplasm for further breeding of drought tolerance in rapeseed.

Creating a homologous restorer line for Ogura cytoplasmic male sterility (Ogu-CMS) in Brassica napus is meaningful for the wider application of Ogu-CMS system in rapeseed production. Previously, an independent development of a new Ogu-CMS restorer line (CLR650) was reported locally from crossing between Raphanobrassica (AACCRR, 2n = 56) and B. napus and a new version of Ogu CMS lines CLR6430 derived from CLR650 was characterized in this study. The results showed that the fertility restoration gene in CLR6430 presented a distorted segregation in different segregating populations. However, the majority of somatic cells from roots had a regular chromosome number (2n = 38) and no radish signal covered a whole chromosome was detected using GISH. Thirty-two specific markers derived from the introgressed radish fragments were developed based on the re-sequencing results. Unique radish insertions and differences between CLR6430 and R2000 were also identified through both radish-derived markers and PCR product sequences. Further investigations on the genetic behaviors, interactions between the fertility restoration and other traits and specific molecular markers to the introgression in CLR6430 were also conducted in this study. These results should provide the evidence of nucleotide differences between CLR6430 and R2000, and the specific markers will be helpful for breeding new Ogura restore lines in future.

Abstract Background Accumulation of storage compounds during seed development plays an important role in the life cycle of oilseed plants; these compounds provide carbon and energy resources to support the establishment of seedlings. Results In this study, we show that BnCIPK9 has a broad expression pattern in Brassica napus L. tissues and that wounding stress strongly induces its expression. The overexpression of BnCIPK9 during seed development reduced oil synthesis in transgenic B. napus compared to that observed in wild-type (WT) plants. Functional analysis revealed that seed oil content (OC) of complementation lines was similar to that of WT plants, whereas OC in Arabidopsis thaliana (L.) Heynh. Atcipk9 knockout mutants (cipk9) was higher than that of WT plants. Seedling of cipk9 mutants failed to establish roots on a sugar-free medium, but root establishment could be rescued by supplementation of sucrose or glucose. The phenotype of complementation transgenic lines was similar to that of WT plants when grown on sugar-free medium. Mutants, cipk9, cbl2, and cbl3 presented similar phenotypes, suggesting that CIPK9, CBL2, and CBL3 might work together and play similar roles in root establishment under sugar-free condition. Conclusion This study showed that BnCIPK9 and AtCIPK9 encode a protein kinase that is involved in sugar-related response and plays important roles in the regulation of energy reserves. Our results suggest that AtCIPK9 negatively regulates lipid accumulation and has a significant effect on early seedling establishment in A. thaliana. The functional characterization of CIPK9 provides insights into the regulation of OC, and might be used for improving OC in B. napus. We believe that our study makes a significant contribution to the literature because it provides information on how CIPKs coordinate stress regulation and energy signaling.

Most flowering plants have evolved a self-incompatibility (SI) system to maintain genetic diversity by preventing self-pollination. The Brassica species possesses sporophytic self-incompatibility (SSI), which is controlled by the pollen- and stigma-determinant factors SP11/SCR and SRK. However, the mysterious molecular mechanism of SI remains largely unknown. Here, a new class II S haplotype, named BrS-325, was identified in a pak choi line ‘325’, which was responsible for the completely self-compatible phenotype. To obtain the entire S locus sequences, a complete pak choi genome was gained through Nanopore sequencing and de novo assembly, which provided a good reference genome for breeding and molecular research in B. rapa. S locus comparative analysis showed that the closest relatives to BrS-325 was BrS-60, and high sequence polymorphism existed in the S locus. Meanwhile, two duplicated SRKs (BrSRK-325a and BrSRK-325b) were distributed in the BrS-325 locus with opposite transcription directions. BrSRK-325b and BrSCR-325 were expressed normally at the transcriptional level. The multiple sequence alignment of SCRs and SRKs in class II S haplotypes showed that a number of amino acid variations were present in the contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I and HV II) of BrSRK-325s, which may influence the binding and interaction between the ligand and the receptor. Thus, these results suggested that amino acid variations in contact sites may lead to the SI destruction of a new class II S haplotype BrS-325 in B. rapa. The complete SC phenotype of ‘325’ showed the potential for practical breeding application value in B. rapa.

Temperature-sensitive male sterility is a heritable agronomic trait affected by genotype-environment interactions. In rapeseed (Brassica napus), Polima (pol) temperature-sensitive cytoplasmic male sterility (TCMS) is commonly used for two-line breeding, as the fertility of pol TCMS lines can be partially restored at certain temperatures. However, little is known about the underlying molecular mechanism that controls fertility restoration. Therefore, we aimed to investigate the fertility conversion mechanism of the pol TCMS line at two different ambient temperatures (16 °C and 25 °C). Our results showed that the anthers developed and produced vigorous pollen at 16 °C but not at 25 °C. In addition, we identified a novel co-transcript of orf224-atp6 in the mitochondria that might lead to fertility conversion of the pol TCMS line. RNA-seq analysis showed that 1637 genes were significantly differentially expressed in the fertile flowers of 596-L when compared to the sterile flower of 1318 and 596-H. Detailed analysis revealed that differentially expressed genes were involved in temperature response, ROS accumulation, anther development, and mitochondrial function. Single-molecule long-read isoform sequencing combined with RNA sequencing revealed numerous genes produce alternative splicing transcripts at high temperatures. Here, we also found that alternative oxidase, type II NAD(P)H dehydrogenases, and transcription factor Hsfs might play a crucial role in male fertility under the low-temperature condition. RNA sequencing and bulked segregant analysis coupled with whole-genome sequencing identified the candidate genes involved in the post-transcriptional modification of orf224. Overall, our study described a putative mechanism of fertility restoration in a pol TCMS line controlled by ambient temperature that might help utilise TCMS in the two-line breeding of Brassica crops.

Self-incompatibility (SI) is a pollen-stigma recognition system controlled by a single and highly polymorphic genetic locus known as the S-locus. The S-locus exists in all Brassica napus (B. napus, AACC), but natural B. napus accessions are self-compatible. About 100 and 50 S haplotypes exist in Brassica rapa (AA) and Brassica oleracea (CC), respectively. However, S haplotypes have not been detected in B. napus populations. In this study, we detected the S haplotype distribution in B. napus and ascertained the function of a common S haplotype BnS-6 through genetic transformation. BnS-1/BnS-6 and BnS-7/BnS-6 were the main S haplotypes in 523 B. napus cultivars and inbred lines. The expression of SRK in different S haplotypes was normal (the expression of SCR in the A subgenome affected the SI phenotype) while the expression of BnSCR-6 in the C subgenome had no correlation with the SI phenotype in B. napus. The BnSCR-6 protein in BnSCR-6 overexpressed lines was functional, but the self-compatibility of overexpressed lines did not change. The low expression of BnSCR-6 could be a reason for the inactivation of BnS-6 in the SI response of B. napus. This study lays a foundation for research on the self-compatibility mechanism and the SI-related breeding in B. napus.

Despite heterosis contributing to genetic improvements in crops, root growth heterosis in rapeseed plants is poorly understood at the molecular level. The current study was performed to discover key differentially expressed genes (DEGs) related to heterosis in two hybrids with contrasting root growth performance (FO; high hybrid and FV; low hybrid) based on analysis of the root heterosis effect. Based on comparative transcriptomic analysis, we believe that the overdominance at the gene expression level plays a critical role in hybrid roots’ early biomass heterosis. Our findings imply that a considerable increase in up-regulation of gene expression underpins heterosis. In the FO hybrid, high expression of DEGs overdominant in the starch/sucrose and galactose metabolic pathways revealed a link between hybrid vigor and root growth. DEGs linked to auxin, cytokinin, brassinosteroids, ethylene, and abscisic acid were also specified, showing that these hormones may enhance mechanisms of root growth and the development in the FO hybrid. Moreover, transcription factors such as MYB, ERF, bHLH, NAC, bZIP, and WRKY are thought to control downstream genes involved in root growth. Overall, this is the first study to provide a better understanding related to the regulation of the molecular mechanism of heterosis, which assists in rapeseed growth and yield improvement.

Timely flowering is important for seed formation and maximization of rapeseed (Brassica napus) yield. Here, we performed flowering-time quantitative trait loci (QTL) mapping using a double haploid (DH) population grown in three environments to study the genetic architecture. Brassica 60 K Illumina Infinium™ single nucleotide polymorphism (SNP) array and simple sequence repeat (SSR) markers were used for genotyping of the DH population, and a high-density genetic linkage map was constructed. QTL analysis of flowering time from the three environments revealed five consensus QTLs, including two major QTLs. A major QTL located on chromosome A03 was detected specifically in the semi-winter rapeseed growing region, and the one on chromosome C08 was detected in all environments. Ribonucleic acid sequencing (RNA-seq) was performed on the parents’ leaves at seven time-points in a day to determine differentially expressed genes (DEGs). The biological processes and pathways with significant enrichment of DEGs were obtained. The DEGs in the QTL intervals were analyzed, and four flowering time-related candidate genes were found. These results lay a foundation for the genetic regulation of rapeseed flowering time and create a rapeseed gene expression library for seven time-points in a day.

Abstract As a desirable agricultural trait, multilocular trait of rapeseed (Brassica rapa; Brassica napus; Brassica juncea), always represents higher yield per plant compared with bilocular plants. We previously isolated a trilocular gene locus, Bjmc1, and identified a set of molecular markers linked to the trilocular gene. With a map-based cloning, we identified that the BjMc1 was located in B genome of Brassica juncea, and it was a CLAVATA1 (CLV1) gene homologue. The insertion of a copia-LTR retrotransposable element 1 (RTE1) into the coding region of BjMc1 interrupted its transcription in rapeseed, leading to the trilocular phenotype. Phylogenetic analysis showed that Mc1 genes were conserved and widespread in land plants. Two amino acid sites had undergone positive selection in the ancestor of Mc1 genes, and then purifying selection was the dominant force after the divergence of dicots and monocots from their common ancestor in the evolutionary process, indicating that Mc1 genes are conserved in modern land plants. Our results provided new insights in molecular regulatory mechanism of multilocularity in rapeseed, and better understanding of molecular mechanism in crop yield improvement.

Monoacylglycerol lipase (MAGL) hydrolyzes monoacylglycerol, producing free fatty acid and glycerol. Although this enzyme has been shown to play important roles in mammal, its potential function in plants remains poorly understood. In a survey of the MAGL genes in Brassica napus, we found tapetal expression of BnaC.MAGL8.a, a homolog of AtMAGL8, results in male sterility in Arabidopsis thaliana. Retarded tapetal PCD and defective pollen wall were observed in the transgenic plants. The tapetal cells became vacuolated at stage 9, and then degenerated at stage 11. Most microspores degenerated with the tapetal cells, and only few pollen grains with an irregular-shaped exine layer were produced in the transgenic plants. Transcriptome analysis identified 398 differentially expressed genes. Most of them are involved in pollen development and stress response. ABORTED MICROSPORES and its downstream pollen wall biosynthesis genes were down-regulated, but genes related with reactive oxygen species homeostasis and jasmonates signaling were up-regulated in the transgenic plants. These results suggest that expression of BnaC.MAGL8.a in tapetum invokes stress response and impairs pollen development. The apparent phenotypic similarity between atgpat1 mutant and BnA9::BnaC.MAGL8.a transgenic plants lead us to propose a role for monoacylglycerol (MAG) in pollen development in Arabidopsis. Our study provides insights on not only the biological function of plant MAGL genes but also the role of MAG in pollen development.

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.

The peanut (Arachis hypogaea L.) is an important grain legume extensively cultivated worldwide, supplying edible oil and protein for human consumption. As in many other crops, plant height is a crucial factor in determining peanut architecture traits and has a unique effect on resistance to lodging and efficiency of mechanized harvesting as well as yield. Currently, the genetic basis underlying plant height remains unclear in peanut, which have hampered marker-assisted selection in breeding. In this study, we conducted a quantitative trait locus (QTL) analysis for peanut plant height by using two recombinant inbred line (RIL) populations including “Yuanza 9102 × Xuzhou 68-4 (YX)” and “Xuhua 13 × Zhonghua 6 (XZ)”. In the YX population, 38 QTLs including 10 major QTLs from 9 chromosomes were detected in 4 environments, and 8 consensus QTLs integrated by meta-analysis expressed stably across multiple environments. In the XZ population, 3 major QTLs and seven minor QTLs from 6 chromosomes were detected across 3 environments. Generally, most major QTLs from the two populations were located on pseudomolecule chromosome 9 of Arachis duranesis (A09), indicating there would be key genes on A09 controlling plant height. Further analysis revealed that qPHA09.1a from the XZ population and one consensus QTL, cqPHA09.d from the YX population were co-localized in a reliable 3.4 Mb physical interval on A09, which harbored 161 genes including transcription factors and enzymes related to signaling transduction and cell wall formation. The major and stable QTLs identified in this study may be useful for further gene cloning and identification of molecular markers applicable for breeding.

Cadmium is a potentially toxic heavy metal to human health. Rapeseed (Brassica napus L.), a vegetable and oilseed crop, might also be a Cd hyperaccumulator, but there is little information on this trait in rapeseed. We evaluated Cd accumulation in different oilseed accessions and employed a genome-wide association study to identify quantitative trait loci (QTLs) related to Cd accumulation. A total of 419 B. napus accessions and inbred lines were genotyped with a 60K Illumina Infinium SNP array of Brassica. Wide genotypic variations in Cd concentration and translocation were found. Twenty-five QTLs integrated with 98 single-nucleotide polymorphisms (SNPs) located at 15 chromosomes were associated with Cd accumulation traits. These QTLs explained 3.49–7.57% of the phenotypic variation observed. Thirty-two candidate genes were identified in these genomic regions, and they were 0.33–497.97 kb away from the SNPs. We found orthologs of Arabidopsis thaliana located near the significant SNPs on the B. napus genome, including NRAMP6 (natural resistance-associated macrophage protein 6), IRT1 (iron-regulated transporter 1), CAD1 (cadmium-sensitive 1), and PCS2 (phytochelatin synthase 2). Of them, four candidate genes were verified by qRT-PCR, the expression levels of which were significantly higher after exposure to Cd than in the controls. Our results might facilitate the study of the genetic basis of Cd accumulation and the cloning of candidate Cd accumulation genes, which could be used to help reduce Cd levels in edible plant parts and/or create more efficient hyperaccumulators.

Small peptides secreted to the extracellular matrix control many aspects of the plant’s physiological activities which were identified in Arabidopsis thaliana, called ATSPs. Here, we isolated and characterized the small peptide gene Bna.SP6 from Brassica napus. The BnaC.SP6 promoter was cloned and identified. Promoter deletion analysis suggested that the -447 to -375 and -210 to -135 regions are crucial for the silique septum and pollen expression of BnaC.SP6, respectively. Furthermore, the minimal promoter region of p158 (-210 to -52) was sufficient for driving gene expression specifically in pollen and highly conserved in Brassica species. In addition, BnaA.bZIP1 was predominantly expressed in anthers where BnaC.SP6 was also expressed, and was localized to the nuclei. BnaA.bZIP1 possessed transcriptional activation activity in yeast and protoplast system. It could specifically bind to the C-box in p158 in vitro, and negatively regulate p158 activity in vivo. BnaA.bZIP1 functions as a transcriptional repressor of BnaC.SP6 in pollen activity. These results provide novel insight into the transcriptional regulation of BnaC.SP6 in pollen activity and the pollen/anther-specific promoter regions of BnaC.SP6 may have their potential agricultural application for new male sterility line generation.

Drought stress is one of the major abiotic factors affecting Brassica napus (B. napus) productivity. In order to identify genes of potential importance to drought stress and obtain a deeper understanding of the molecular mechanisms regarding the responses of B. napus to dehydration stress, we performed large-scale transcriptome sequencing of B. napus plants under dehydration stress using the Illumina sequencing technology. In this work, a relatively drought tolerant B. napus line, Q2, identified in our previous study, was used. Four cDNA libraries constructed from mRNAs of control and dehydration-treated root and leaf were sequenced by Illumina technology. A total of 6018 and 5377 differentially expressed genes (DEGs) were identified in root and leaf. In addition, 1745 genes exhibited a coordinated expression profile between the two tissues under drought stress, 1289 (approximately 74%) of which showed an inverse relationship, demonstrating different regulation patterns between the root and leaf. The gene ontology (GO) enrichment test indicated that up-regulated genes in root were mostly involved in “stimulus” “stress” biological process, and activated genes in leaf mainly functioned in “cell” “cell part” components. Furthermore, a comparative network related to plant hormone signal transduction and AREB/ABF, AP2/EREBP, NAC, WRKY and MYC/MYB transcription factors (TFs) provided a view of different stress tolerance mechanisms between root and leaf. Some of the DEGs identified may be candidates for future research aimed at detecting drought-responsive genes and will be useful for understanding the molecular mechanisms of drought tolerance in root and leaf of B. napus.